Abstract
BackgroundRSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.ResultsNovel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range.ConclusionNovel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.
Highlights
RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants
ΛRed-mediated substitution of RSF1010 mob locus by an in vitro generated DNA fragment containing an auto-regulated element PlacUV5→lacI marked by chloramphenicol resistance gene and the flanks strictly homologous to the target sites on the plasmid, was performed (Fig. 2) and described in detail in Methods
The resultant bi-plasmid strain grown in arabinose-containing medium for induction of λRed genes was electroporated with the constructed DNA fragment
Summary
RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Hundreds of bacterial genomes are available for comparative genomics. The vast genetic information as well as introduction of new bacterial strains to industrial usage require the suitable tools for rapid genetic manipulations with a wide variety of bacterial species. Broad-host-range plasmids provide a valuable tool for such work. Numerous broad-host-range shuttle vectors, promoter-probe vectors, expression vectors and other special-purpose vectors are available now. The majority of them are based on the replicons of the IncQ or IncP groups [see for example [1,2,3]]
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